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PROTEIN FOLDING
Protein folding: the Levinthal Paradox (1968)
Assuming 3 possible conformations for each residue
An interconversion time of 10-12 sec
For a 150-residue protein: 3150 = 1068 possible conformations
would be explored after 1056 (1046) years!!!! (more than the
age of the universe)...and proteins fold in a few miliseconds!
Levinthal’s proposal: There SHOULD be a defined folding
pathway through a defined series of intermediates, instead of
a fully random process
Are there pathways for protein folding?
C. Levinthal, J. Chem. Phys. 65, 44 (1968).
Unfolded
Native
K.A. Dill & H.S. Chan, Nature Struct. Biol. 4, 4 (1997).
Levinthal Paradox:
How do proteins find the native “needle” in the conformational
“haystack” in milliseconds when the free energy bias toward the
native state is so small?
Folding Problem
• What structure does a polypeptide fold into?
• Unfolded states expose hydrophobic side chains that
should be hidden from water, intermolecular interactions
of hydrophobic side chains leads to aggregation /
precipitation, consequence can be misfolding diseases.
• Unfolded states are prone to be digested by proteases.
• On the ribosome: How does a 1/2-formed protein chain
behave?
First report of protein refolding in vitro
Anson and Mirsky
Alkali-denatured hemoglobin spontaneously recover its
biological properties when the pH is returned to
neutrality.
Anson, M.L. and Mirsky, A. E. (1925) J. Physiol. 60, 50-67.
Renaturation of
unfolded,
denatured
ribonuclease
C. Anfinsen,
1972 Nobel
Laureate
The amino acid sequence of
a polypeptide chain contains
all the information required
to fold the chain into its
native, 3D structure.
Anfinsen, C. B., Haber, E., Sela, M. and White, F. H. (1961)
Proc. Natl. Acad. Sci. USA 47, 1309-1314.
Sequence determines fold, but....
Protein Folding Related
Diseases
Natively Unfolded Proteins
Why study folding?
• What makes a protein stable: kinetic
stability or thermodynamic stability?
• If we design a new protein, will it fold?
• Protein production by overexpression
• Protein stabilization by rational
mutagenesis
• To understand misfolding diseases
Flexibility vs. Stability trade-off
DePristo, Weinreich & Hartl, Nature Rev Gen. (2005)
Thermodynamics of Protein
Folding
ΔG = ΔH − T Δ S
ΔG = RT ln K
ΔG fold = ΔGnative − ΔGdenatured
ΔGfold is small, about –5 to -20 kcal/mol < kT/10/aa !!!!!
therefore the ratio of unfold/fold varies between 0.13 to 0.0003
•Comes from a near balance of opposing large forces
•Small forces can therefore play an important role
•“Native” state is global minimum of ΔGfold
•Folding is reversible (Anfinsen, 1973)
The forces that drive folding
Reversibility implies ΔGfold is minimized
ΔG composed of many contributions
• Hydrophobic exclusion (kj/mol) entropic
stabilizing
• Electrostatics: hydrogen bonding and salt bridges (j/mol)
+/-
• entropy (degrees of freedom, flexibility) (kj/mol)
destabilizing
• solvation of polar and charged residues (kj/mol)
stabilizing
• steric repulsion (kj/mol)
destabilizing
• van der Waal’s interactions (j/mol)
stabilizing
Structure and Mechanism in protein science
Alan Fersht
Energetics of the 3D Structure
Experimental Approach: to introduce mutations on a known structure
and then monitor stability changes
Ala mutations: Smallest possible side chain
Gly mutations: to explore flexibility and allowed conformations
α-Helix formation
• High entropy
• Conformational freedom
• Reduced steric contacts
• Conformational freedom is lost
• Intrahelical H bonds entropically favored
• Hydrophobic burial
Energetics of the 3D Structure
β-Sheet formation
• Driven by hydrophobic interactions between side chains
• β-Branched residues (Thr, Ala) may be favored due to larger degrees
of freedom in extended strands
• Gly: disfavored
(Un)folding experiments
• A protein isolated from an organism usually
has a well-defined 3D structure (fold).
• Changing conditions can unfold the protein:
- heat or pressure denaturation
- pH denaturation
- denaturation by chemicals
• Often, but not always, the protein can refold if one takes away the denaturant.
• Then, we study the folding transition
(kinetics & thermodynamics)
Unfolded state
• Is the conformation of unfolded proteins
random, or is there residual native
structure or other structure? We don’t
know.
• We have a static picture of the F state,
don’t know if that is appropriate: Some
motions within F must be the motions that
can lead to unfolding.
• Do different denaturants induce the same
unfolded state, or is it U, U’, U’’ … ?
What stabilises the fold?
• Often a mutation destabilises G only a little
• Conclusion: Many interactions add small stability
increments.
• Isolated helices of proteins don’t fold, or the fold is
short-lived, because all interactions added ~kT.
• The minimum peptide length that can fold is ~50
amino acids.
• But sometimes a few interactions are crucial.
• A mutation can have different effects on stability and
on folding kinetics!
To study protein folding, we need to use different probes
To study protein folding, we need to use different probes
Es una
Serpiente ?
Hmmm..
Que rico
Purecito!!!
Es una
Manguera?
Es un
Arbol?
Es una
Alfombra?
Yeh & Rousseau (2000) Nature Structural Biology 7, 443-445.
Table 1. Methods used to investigate protein folding and aggregation
Continued
Two state model
Two-state transition?
U (unfolded) ⇔ N (native)
Two state-model:
• No intermediate accumulation
• Cooperative process
Intermediates at low concentration may not be detected,
giving rise to apparent two-state transitions.
U: Unfolded states (ensemble of molecules)
May be characterized by IR, CD, NMR, SAXS,
hydrodynamics measurements
Observation of partially folded species
• H-bonded HN in secondary
structures of folded
proteins exchange slowly
with D2O.
• Flexible loops get Dlablelled quickly.
• If a protein unfolds partly,
the HN that disappear fast
in D2O belong to the
unfolded part.
Partially folded species
• Some proteins can form partly folded
states.
• For example, in acid-denatured α
lactalbumin, helices B and C are protected
from HN exchange.
• This means there a partially folded
structure can be formed, but does this
structure occur during folding?
The molten globule
•
•
•
•
•
•
•
Intermediate found in Compact structure
Partially folded
May be isolated in some cases
Few tertiary contacts
Native Secondary structure is retained
Fluctuating hydrophobic core
There is an energetic barrier to reach the Native state
The molten globule
Lactalbumin at pH 2 is a molten globule
The molten globule
The radius of gyration of native α-lactalbumin is 15.7
Å, but the acid molten globule has a radius of 17.2 Å.
Molten globule α-lactalbumin retains a globular
shape, but is simply ‘swollen’ from the native state
(containing 270 bound water molecules).
Lactalbumin at pH 2 is a molten globule
The molten globule
Lactalbumin at pH 2
The molten globule
Lactalbumin at pH 2 (molten globule)
NH signals exchange faster than in
the native state
Timescales of experiments for studying
protein folding
Bieri and Kiefhber, Biol. Chem., 1999
Stopped Flow: how does it
work?
Excitation
Fluo.
Cell
Mixer
A
Stopping
Syringe
B
DRIVE
Standard stopped flow schematic
z Under pneumatic drive activation, the
two small volumes of solutions are
driven from high performance syringes
through a high efficiency mixer.
z The resultant mixture passes through a
measurement flow cell and into a
stopping syringe.
z Just prior to stopping, a steady state
flow is achieved.
z As the solution fills the stopping syringe,
the plunger hits a block, causing the
flow to be stopped instantaneously.
z Using appropriate techniques, the
kinetics of the reaction can be
measured in the cell.
A(t) =ΣAi exp(-kit) + A∞
where A(t) is the amplitude of the change at time t, A∞ is
the amplitude at infinite time, Ai is the amplitude at zero
time of phase i, and ki is the rate of phase i.
For 1-cm window length, 10 m/s flow rate
observation time = 0.01/10 = 0.001 s = 1 ms
Methods to Study Folding: NMR
Folding of Lysozyme
Folding models / pathway
• What structures form when during folding?
• Nucleation / growth: one secondary structure element
forms first, then rest attaches to this
• Diffusion-collison: all secondary structure form first,
then form tertiary structure
• Hydrophobic collapse: whole protein become
compact first, then secondary structures form
• Jigsaw: If a protein folds starting from very different U
states, does it fold along the same same path?
Figure 19.1 from Fersht
Energetics and Kinetics
Energetics and Kinetics
Energetics and Kinetics
The Pathway Solution
The bottom of the funnel is thermodynamic minimum
Many different kinetic paths reach the bottom
Energetics and Kinetics
Energetics and Kinetics
Protein Folding Funnel
Peter Leopold and Jose
Onuchic introduced the
protein folding funnel
approach to the problem.
(PNAS 1992)
Q=ratio of native contact
What do we mean by “folded” protein?
The folded state is a collection of conformers that rapidly
interconvert since the energy barrier is low at room temp.
These energy barriers are small.
Structure of the Transition State
Solution to the Levinthal
Paradox
The essence of the solution to the ‘Levinthal Paradox’ that has emerged
from the lattice simulations is that an individual molecule needs to sample only
a very small number of conformations because the nature of the effective
energy surface restricts the search and there are many transition states.
Dinner et al., TIBS, 2000, 25, p.331
More Complex Proteins = More
Complex Folding: Lysozyme
20%
70%
Rapid collapse
Qα native contacts in α domain
Qβ native contacts in β domain